Structure/function and mode of action of antimicrobial

Antimicrobial cationic peptides are ubiquitous in nature and are thought to be a component of the first line of defense against infectious agents. It is important to study these peptides in order to use them as potential templates for new antibiotic therapeutics. The aim of this study was to determine the structure/activity relationships of selected peptides and to determine the mode of action of these peptides on Gram positive bacteria. Studies with model membrane systems using circular dichroism and fluorescence spectroscopy indicated that these active peptides were induced into a more defined structure upon binding to lipids and detergents. In general, peptide interaction with lipids and detergents were similar. The peptides entered a hydrophobic environment, with tryptophan residues inaccessible to the aqueous solution. Although there were differences between peptides in specific interactions, no correlation could be made between lipid interaction and antimicrobial specificity or activity. The activity of cationic peptides from different structural classes was determined on various Gram positive strains and the killing kinetics of these peptides were very similar at 10-fold the MIC. Electron microscopy of S. aureus and S. epidermidis treated with the peptides at 10-fold the MIC showed variability in effects on bacterial structure. Mesosome-like structures were seen to develop in S. aureus with all peptides, whereas different effects, including nuclear condensation, were seen in the case of S. epidermidis. The membrane-potential-sensitive fluorophore DiSC₃(5) was utilized to assess the interaction of antimicrobial peptides with the cytoplasmic membrane of S. aureus. Studies of the kinetics of killing and membrane depolarization showed that no correlation could be made between cytoplasmic membrane depolarization and peptide activity. Thus, although cytoplasmic membrane permeabilization was a widespread ability among peptides, it did not appear to be the killing mechanism. Macromolecular synthesis assays showed that all peptides studied had intracellular effects, and these were often seen at sub-lethal peptide concentrations. The peptides differed in their specific effects on macromolecular synthesis. In general, evidence presented here suggested that cytoplasmic membrane disruption is not the sole mechanism of action against bacteria, and that multiple targets may be involved. The specific mechanism differs between peptides of different structural classesScience, Faculty ofMicrobiology and Immunology, Department ofGraduat